The long-term objective of this project is to elucidate the molecular mechanisms controlling the assembly of icosahedral viral capsids. The assembly of icosahedral viruses if pathway dependent, however, the mechanism by which the pathway is encoded within the protein subunits is unknown. It has been suggested that energetic differences between assembly intermediates determines the assembly pathway. This project will test that hypothesis by taking advantage of the fact hat the assembly of procapsid of the dsDNA containing P22 bacteriophage requires the activity of scaffolding protein, and that scaffolding protein is likely to act by increasing the relative stabilities of assembly intermediates which lie along the pathways to a T=7 procapsid lattice. The approach will be to take advantage of the modular nature of scaffolding protein to selectively alter the key determinants of scaffolding action and evaluate the effect of assembly. These efforts will culminate in the key determinants of scaffolding action, and evaluate the effect on assembly. These efforts will culminate in the redesign of scaffolding protein to promote the assembly of T=4 and T=13, rather than T=7 capsids. The specific aims are to 1) determine the relative contribution of individual amino acids to coat/scaffolding binding; 2) localize the dimeric and tetrameric interfaces in the scaffolding protein to enable structural analysis, 3) evaluate the role of oligomerization and the spatial orientation of coat protein domains in assembly, and 4) reprogram the scaffolding protein to make T=4 and T=13 capsids. Upon completion of this project, a detailed quantitative description of the role of scaffolding protein in controlling viral assembly pathways by altering the relative stabilities of assembly intermediates will be available. This will make possible a critical evaluation of the hypothesis that, in general, capsid assembly pathways are controlled by the relative stabilities of intermediates. Furthermore, a quantitative description of the control of assembly is critical to the consideration of anti-viral therapeutics targeted at subunit interactions during assembly in general. In particular, both herpes and adenovirus require the action of scaffolding protein for assembly, and interfering with scaffolding protein function represents a viable antiviral approach for these viruses. Finally, the increasing research efforts devoted to the development of viruses as delivery vehicles for gene therapy applications suggests that the ability to alter the assembly pathway of virions in a controlled fashion will ultimately be essential.